Astaxanthin is a naturally occurring carotenoid which is responsible for the red colour in salmon, trout, sea bream and other fishes. In natural surroundings this carotenoid is obtained with the feed of these animals in the form of crustaceans and other astaxanthin-containing organisms.
Fish raised on fish-farms or in hatcheries are generally pale and lack the skin and flesh colours of their naturally raised congeners, this is due to a lack of dietary astaxanthin. The addition of astaxanthin to the feed of the fishes is an appropriate way to overcome the colour problem of fish raised in non-natural surroundings.
Astaxanthin applied for this purpose can be obtained by semi-synthetical means. For regulatory reasons and due to increasing pressure excercised by consumers it may be favourable to use astaxanthin from natural sources.
Natural sources of astaxanthin are krill and crawfish shells, algae, flowers and yeast. Astaxanthin extracted from krill and crawfish is very expensive. The yield of astaxanthin in algae is high but large-scale production of algae is difficult. Therefore attention has turned to the use of yeast, specifically Phaffia rhodozyma for the production of astaxanthin.
The astaxanthin-containing yeast Phaffia rhodozyma was first isolated in the early 1970s from exudates of deciduous trees in mountainous regions of Japan and Alaska (Phaff et al. (1972) in Proceedings of the 4th IFS: Fermentation Technology Today, p. 759-774. Ed. G. Terui, Kyoto). In wild-type Phaffia rhodozyma strains astaxanthin concentrations have been determined. Johnson and Lewis ((1979), J. Gen. Microbiol. 155 173-183) have found that the concentration of astaxanthin which is dependent on culture conditions varies considerably but never exceeds 650 .mu.g per g yeast dry weight. When growing the yeast under different growth conditions the authors have found, after acetone extraction of the carotenoids, that one of the main contaminants of astaxanthin is 3-hydroxy-3',4'-didehydro-.beta.,.psi.-caroten-4-one (HDCO).
Under aerobic conditions the amount of HDCO is about 0.5-1.5% of the amount of astaxanthin. Under microaerophilic conditions this amount increases up to about 26%, (Johnson and Lewis cited above) this amount is too high.
The astaxanthin concentration of about 650 .mu.g/g dry weight as reported for wild type Phaffia rhodozyma strains is much too low to make the process economically attractive. Therefore extensive studies have been performed to increase the yield of astaxanthin.
As indicated above Johnson and Lewis (1979) already describe a myriad of different culture conditions (pH, temperature, carbon source, oxygen pressure and light are but a number of factors) which influence the intracellular astaxanthin concentration in Phaffia. A more promising approach for obtaining higher astaxanthin concentrations is classical mutagenesis. An and Johnson ((1990) Antonie van Leeuwenhoek 57: 191-203) report that N-methyl-N'-nitro-N -nitrosoguanidine (NTG) mutagenesis of natural Phaffia rhodozyma strains gave rise to strains with increased carotenoid contents, amounts of up to 1050 .mu.g/g dry weight are reported.
The strains grown under different conditions described by An and Johnson (opt.cited) were found to contain from 10-15% of total carotenoids in the form of HDCO (about 30% of the amount of astaxanthin). Also Lewis et al ((1990) Appl. Environm. Microbiol. 56: 2944-2945) have shown that the amount of HDCO as compared to astaxanthin increases due to mutagenesis. In their situation (Table 1) HDCO increased from 4 to 8%.
No biological effects of HDCO are known. In fact till now HDCO has only been reported in Phaffia rhodozyma. High amounts of HDCO which seem to be increasing due to mutagenesis are unacceptable as additions to animal feed.
Therefore there is clearly a need for astaxanthin obtained from Phaffia which has a low HDCO content. The present invention provides such an astaxanthin.